Recombinant adenovirus (rAd) vectors have desirable features for gene delivery, including wide tissue and cell tropism, the capacity to accommodate large expression cassettes and high transduction efficiency. In addition, adenovirus is well suited for pharmaceutical development as the virus grows to high specific titers and scalable manufacturing processes have been established (Huyghe et al., (1995) Hum. Gene Ther. 6:1403-1416; Shabram et al., (1997) Hum. Gene Ther. 8:453-465). Production of rAd vectors requires engineered cell lines that can complement functions removed from the viral genome. For pharmaceutical development and commercial manufacture of viral vectors, the vector-cell line combination also must be amenable to scale-up and provide material of sufficient quality and purity.
Replication defective rAd vectors for gene therapy use are generally deleted for the viral early region 1 (E1). E1 contains two transcription units, E1A and E1B, which encode a number of proteins that have critical roles in the early and late phases of the lytic cycle. Production of rAd vectors requires complementation of these E1 activities. E1A and E1B gene functions have been extensively characterized. A review of E1A and E1B functions is provided in Bayley, et al. (1994) International J. of Oncology 5:425-444 and Shenk, et al. (1996) in Adenoviridae: The Viruses and Their Replication, Fields Virology (K. D. M. Fields B. N., and Howley, P. M., Ed.), 2 Lippincott-Raven, Philadelphia, Pa. The first E1-complementing line, 293, was generated by transfection of primary embryonic kidney cells using physically sheared adenovirus 5 DNA (Graham et al., (1977) J Gen. Virol. 36:59-74). Genomic analysis subsequently demonstrated that 293 cells carry an integrated fragment of the left-hand end of adenovirus genome (bases 1-4344), containing the E1 region and additional flanking sequences (Louis et al., (1997) Virology 233:423-429).
Although 293 cells produce E1-deficient rAd vectors at acceptable levels, an undesirable contaminant called replication competent adenovirus (“RCA”) is sometimes generated by homologous recombination between the rAd vector and the adenovirus sequences present in the 293 genome (Lochmuller et al, (1994) Hum. Gene Ther. 5:1485-1491; Zhu et al., (1999) Hum. Gene Ther. 10:113-121). To reduce the risk of generation of RCA by homologous recombination, Fallaux et al., (Hum. Gene Ther. 9:1909-1917 (1998)) transfected human embryonic retinoblasts with a recombinant plasmid containing E1 genes, in which the E1A promoter and E1B polyadenylation sequences were replaced by heterologous control elements. Deletion of the adenovirus flanking sequences in the E1 plasmid yielded a cell line, PER.C6, which does not generate RCA through homologous recombination when cell line-matched rAd vectors are employed (Fallaux et al., (1998) Hum. Gene Ther. 9:1909-1917). However, recent studies have shown that an atypical form of RCA, called helper-dependent E1-positive particles, can be formed when non-matched adenoviral vectors are propagated in PER.C6 cells (Murakami et al., (2002) Hum. Gene Ther. 13:1909-1920).
The E1 region used for complementation of E1-deleted adenoviruses in 293 cells and PER.C6 cells includes the entire E1B transcription unit, which encodes two major proteins: E1B-19K and E1B-55K. In adenovirus replication, the E1B-19K and E1B-55K proteins function in the early lytic cycle to limit E1A-induced apoptosis (Querido et al., (1997) J. Virol. 71:3788-3798; Rao et al., (1992) Proc. Natl. Acad. Sci. USA 89:7742-7746; White et al., (1991) J. Virol. 65:2968-2978). In addition, E1B-55K functions in the late phase to stimulate the accumulation and translational of viral late mRNAs (Babiss et al., (1985) Mol. Cell Biol. 5:2552-2558; Harada and Berk, (1999) J. Virol. 73:5333-5344). E1B has also has been shown to collaborate with E1A in transforming primary cells (Branton et al., (1985) Biochim. Biophys. Acta 780:67-94), and specifically protects against E1A sensitization to apoptosis (White et al., (1991) J. Virol. 65:2968-2978).
The construction of stable human cell lines that effectively and efficiently complement replication deficient adenoviral vectors can be difficult. A barrier for developing E1-complementing cell lines is the toxicity associated with high levels of E1A gene product expression. For example, constitutive expression of the E1 proteins, especially E1A, has proven difficult in established cell lines (Imler et al, (1996) Gene Ther. 3:75-84). E1A has been shown to suppress cell growth and induce anoikis (Frisch, (1991) Proc Natl Acad Sci USA 88:9077-9081; Frisch and Mymryk, (2002) Nat Rev Mol Cell Biol 3:441-452; Mymryk et al., (1994) Oncogene 9: 1187-1193; Rao et al., (1992) Proc Natl Acad Sci USA 89:7742-7746). Thus, complementation cell lines, such as those known in the art, that constitutively express E1A proteins may be associated with poor survival rates prior to and/or during adenoviral vector production.
Others have previously used human tumor cell lines, such as the A549 cell line, to develop E1-complementing cell lines. For example, Massie (U.S. Pat. No. 5,891,690) transformed A549 cells with an E1 region expression cassette in which the adenovirus E1A promoter was replaced with the human alpha-actin promoter. However, the yield of an adenovirus gene therapy vector expressing the bacterial beta-galactosidase gene was shown to be lower than from 293 cells. Imler, et al. used regulated induction of E1 as a strategy to avoid toxicity associated with constitutive expression of E1A, allowing generation of rAd producer cell lines based on A549 cells (Imler, et al., (1996) Gene Ther. 3:75-84). Production yields of rAd vectors from these Gal4-inducible producer cell lines were reported to be 5-10 fold lower than from 293 cells (Imler, et al., supra).
Accordingly, there is a need for more efficient recombinant cell lines to produce replication-defective adenoviruses (i.e., adenoviruses containing a deletion of the E1A and E1B coding regions of the adenoviral genome) which have low RCA and with production levels on the order of wild-type adenovirus production from the 293 cell line.